Method For Predicting Recurrence And Progression Of Urothelial Cancer Patient After Treatment

ABSTRACT

The present disclosure relates to a method for predicting a recurrence and a progression of an urothelial cancer patient after a treatment including steps as follows. A urine sample is obtained from a subject. The urine sample is performing a serially centrifugation step to obtain a third precipitate. The third precipitate is resuspended with an extraction solvent to obtain a third mixture, and the third mixture is centrifuged to obtain a fourth supernatant. The fourth supernatant is analyzed by a mass spectrometry to detect whether there is a particular peptide therein.

RELATED APPLICATIONS

The present application is a Divisional Application of the applicationSer. No. 15/343,230, filed Nov. 4, 2016, the entire contents of whichare hereby incorporated herein by reference, which claims priority toTaiwan Application Serial Number 105117855, filed Jun. 6, 2016, which isherein incorporated by reference.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR §1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file“CP-3304-1-US_SequenceListing”, created on Nov. 2, 2016, which is 3,409bytes in size.

BACKGROUND Technical Field

The present disclosure relates to a method for validating an existenceof an exosome and applications thereof. More particularly, the presentdisclosure relates to a method for validating the existence of a urinaryexosome and the applications thereof.

Description of Related Art

An urothelium (also called the transitional epithelium) is the lining onthe inside of the bladder, ureters and urethra, as well as the renalpelvis (the part of the kidney where urine collects). An urothelialcancer (UC) is derived from lesions of the urothelium accounting for themajority of urinary tract tumors, which includes carcinomas of thebladder, ureters, and renal pelvis. The UC is the ninth most commonmalignancy worldwide.

Current diagnoses of the UC rely on a urine cytology, a urography(including intravenous urography (IVU) and CT-urography), and acystoscopy aided by biopsy. Though the urine cytology and the urographyare non-invasive, the sensitivity of these tests varies with the UClocation and grade by more than 30%. The cystoscopy with biopsy offersthe most accurate diagnosis and description of the UC. However, thecystoscopy is expensive and invasive, and it is a painful inspectionmethod for patients. Thus, there is a crucial need for noninvasive,objective, and rapid markers that offer adequate sensitivity andspecificity for surveillance and diagnosis of the UC.

Exosomes are membranous microvesicles, fusions of late endosomes andcell membranes, released by living cells into surrounding biofluids. Thereported diameter of exosomes is between 30 nm and 100 nm. Afterexocytosis of the exosomes, the exosomes can be reabsorbed into thecytoplasm via a lipid rafts mediated endocytosis, a clathrin-dependentendocytosis and a caveolin-dependent endocytosis. Recent studiesreported that the exosomes of cancer cells play an important regulatoryrole on the process of cancer and concentrations of the exosomes can beused for detecting the cancers and stages. Another report also reportsthat urinary exosomes from the patients with high-grade bladder cancercan promote UC cells migration and angiogenesis. However, the currentmethods for validating an existence of the urinary exosome rely on anelectron microscopy, a flow cytometry or a Western immunoblotting. Thesemethods are time-consuming and expensive, thus they can not rapidlyvalidate the existence of the urinary exosome.

SUMMARY

According to one aspect of the present disclosure, a method forvalidating an existence of a urinary exosome includes steps as follows.A sampling step is provided, wherein a urine sample is obtained from asubject. A serially centrifugation step is performed on the urinesample, wherein the serially centrifugation step further includes stepsas follows. The urine sample is centrifuged to obtain a firstsupernatant and a first precipitate. The first precipitate isresuspended with an isolation solution to obtain a first mixture, andthe first mixture is centrifuged to obtain a second supernatant and asecond precipitate. The first supernatant and the second supernatant aremixed to obtain a second mixture, and the second mixture is centrifugedto obtain a third precipitate. Then an extraction step is provided,wherein the third precipitate is resuspended with an extraction solventto obtain a third mixture, the third mixture is centrifuged to obtain afourth supernatant, and the extraction solvent is formic acid and/oracetonitrile. A mass spectrometric analysis step is performed on thefourth supernatant by using a matrix-assisted laser desorptionionization-time of flight mass spectrometry (MALDI-TOF MS), wherein theurinary exosome is existed in the fourth supernatant when at least twopolypeptides of a 3367 m/z polypeptide, a 3441 m/z polypeptide, a 3483m/z polypeptide and a 10884 m/z polypeptide are detected in the fourthsupernatant.

According to another aspect of the present disclosure, a non-invasivemethod for identifying an urothelial cancer includes steps as follows. Asampling step is provided, wherein a urine sample is obtained from asubject. A serially centrifugation step is performed on the urinesample, wherein the serially centrifugation step further includes stepsas follows. The urine sample is centrifuged to obtain a firstsupernatant and a first precipitate. The first precipitate isresuspended with an isolation solution to obtain a first mixture, andthe first mixture is centrifuged to obtain a second supernatant and asecond precipitate. The first supernatant and the second supernatant aremixed to obtain a second mixture, and the second mixture is centrifugedto obtain a third precipitate. Then an extraction step is provided,wherein the third precipitate is resuspended with an extraction solventto obtain a third mixture, the third mixture is centrifuged to obtain afourth supernatant, and the extraction solvent is formic acid and/oracetonitrile. A mass spectrometric analysis step is performed on thefourth supernatant by using a matrix-assisted laser desorptionionization-time of flight mass spectrometry (MALDI-TOF MS) to detectwhether the fourth supernatant has a 5593 m/z polypeptide and/or a 5947m/z polypeptide.

According to yet another aspect of the present disclosure, a method forpredicting a recurrence and a progression of an urothelial cancerpatient after a treatment includes steps as follows. A sampling step isprovided, wherein a urine sample is obtained from a subject. A seriallycentrifugation step is performed on the urine sample, wherein theserially centrifugation step further includes steps as follows. Theurine sample is centrifuged to obtain a first supernatant and a firstprecipitate. The first precipitate is resuspended with an isolationsolution to obtain a first mixture, and the first mixture is centrifugedto obtain a second supernatant and a second precipitate. The firstsupernatant and the second supernatant are mixed to obtain a secondmixture, and the second mixture is centrifuged to obtain a thirdprecipitate. Then an extraction step is provided, wherein the thirdprecipitate is resuspended with an extraction solvent to obtain a thirdmixture, the third mixture is centrifuged to obtain a fourthsupernatant, and the extraction solvent is formic acid and/oracetonitrile. A mass spectrometric analysis step is performed on thefourth supernatant by using a matrix-assisted laser desorptionionization-time of flight mass spectrometry (MALDI-TOF MS), wherein theurothelial cancer patient is determined to have a high recurrence and ahigh progression after the treatment when a 5947 m/z polypeptide isdetected in the fourth supernatant.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The present disclosure can be more fully understood by reading thefollowing detailed description of the embodiment, with reference made tothe accompanying drawings as follows:

FIG. 1 is a flow diagram showing a method for validating an existence ofa urinary exosome according to one embodiment of the present disclosure;

FIG. 2 is analytical results of a Western immunoblotting to detectbiomarkers of a urinary exosome of a subject;

FIG. 3 is a set of micrographs of a transmission electron microscope ofthe urinary exosome of the subject;

FIG. 4A is a protein quantitative results according to one embodiment ofthe present disclosure;

FIG. 4B is analytical result of a sliver stain according to oneembodiment of the present disclosure;

FIGS. 5A to 5D are MALDI-TOF mass spectra according to one embodiment ofthe present disclosure;

FIG. 6 is an analytical result of a 2-D pseudo gel according to oneembodiment of the present disclosure;

FIG. 7 is a flow diagram showing a non-invasive method for identifyingan urothelial cancer according to another embodiment of the presentdisclosure;

FIG. 8 is a set of micrographs of an immunohistochemical staining;

FIG. 9A is a quantification of an IHC score of α-1 antitrypsinexpressions in non-UC tissue specimens and UC tissues specimens detectedby the immunohistochemical staining;

FIG. 9B is the quantification of the IHC score of H2B1 K expressions inthe non-UC tissue specimens and the UC tissues specimens detected by theimmunohistochemical staining;

FIG. 10A is the quantification of the IHC score of the α-1 antitrypsinexpressions in high-grade UC tissue specimens and low-grade UC tissuespecimens detected by the immunohistochemical staining;

FIG. 10B is the quantification of the IHC score of the H2B1K antitrypsinexpressions in high-grade UC tissue specimens and low-grade UC tissuespecimens detected by the immunohistochemical staining;

FIG. 11 is a flow diagram showing a method for predicting a recurrenceand a progression of an urothelial cancer patient after a treatmentaccording to yet another embodiment of the present disclosure;

FIG. 12A is a survival curve showing overall recurrence-free accordingto yet another embodiment of the present disclosure; and

FIG. 12B is the survival curve showing overall progression-freeaccording to yet another embodiment of the present disclosure.

DETAILED DESCRIPTION

A method for validating an existence of a urinary exosome, anon-invasive method for identifying an urothelial cancer and a methodfor predicting a recurrence and a progression of an urothelial cancerpatient after a treatment are provided. An exosome isolated from a urinesample is extracted by formic acid and/or acetonitrile. Then a massspectrometric analysis step is performed by using a matrix-assistedlaser desorption ionization-time of flight mass spectrometry to detectwhether there is a particular peptide therein for validating theexistence of the urinary exosome, identifying the urothelial cancer in asubject and predicting the recurrence and the progression of theurothelial cancer patient after the treatment. The following aredescriptions of the specific terms used in the specification.

The term “neutrophil defensin” is a highly basic cationic polypeptidethat is also referred to as human neutrophil peptide. The neutrophildefensin belongs to a kind of antimicrobial peptides. The neutrophildefensins are abundant in neutrophils, epithelial cells, lining thebronchial tress, genitourinary tract and the urinary exsomes.

The term “S100 calcium-binding protein A9” is also known as MRP14(migration inhibitory factor-related protein 14) or calgranulin B. It isa calcium and zinc binding protein in the S100 protein family, which isa calcium-binding protein secreted by the neutrophils and other cellsand released in inflammation.

The term “α-1 antitrypsin” belongs to a family of serum proteinaseinhibitor, which accounts for more than 90% of the total plasmaproteases. The α-1 antitrypsin can inhibit a variety of serineendopeptidase, such as neutrophil elastase, trypsin, plasma andthrombin. The main function of the α-1 antitrypsin is to protect cellsand organs damaged from proteases and inhibit infections andinflammation for maintaining balance of body environment.

The term “H2B1K (histone H2B)” is one of the most abundantlymonoubiquitinated conjugates in nucleus and is involved intranscriptional control of gene expression and DNA damage response.Levels of ubiquitinated H2B are low in advanced cancers includingbreast, colorectal, lung and parathyroid.

Reference will now be made in detail to the present embodiments of thepresent disclosure, examples of which are illustrated in theaccompanying drawings.

EXAMPLES I. Subjects

This clinical trial program is approved by China Medical University &Hospital Research Ethics Committee, wherein 119 subjects include 129urothelial cancer (UC) patients, 17 urinary tract infection (UTI)patients, 25 prostate cancer (PC) patients and 20 healthy subjects.

Table 1 shows clinical features of the subjects, wherein variables ofall subjects include age and gender, and the variables of the UCpatients further include a tumor location at presentation, a pathologicstage, a pathological grade, a tumor diameter, a number of tumors,surgical methods, a lymphovascular invasion, a lymph node metastasis, achemotherapy and a surgical margin. The progression is defined as adistant metastasis, a superficial progression to a muscle invasion, or acancer-related death. The recurrence is defined as new tumor noted aftera transurethral resection bladder tumor (TURBT), secondary primaries,the progression, or the distant metastasis. In terms of the age andgender as the variables, there are no significant difference among theUC patients, the UTI patients, the PC patients and the healthy subjects(p value is 0.178 and 0.328 respectively). In the UC patients, 48.8% ofpatients have pathological stage pTa/Tis/T1, 71.3% of patients havehigh-grade UC, 79.9% of patients have maximum tumor diameters <3 cm,62.8% of patients have multiple tumors, 27.7% of patients havelymphovascular invasion, 7.8% of patients have lymph node metastasis,and 5.4% of patients have positive surgical margins.

TABLE 1 The clinical features of the subjects Comparison Variable UC UTIPC Normal P value Total, n 129 17 25 20 — Age (yrs) 67.34 ± 11.35 64.41± 7.68 63.65 ± 8.85 69.88 ± 9.96 0.178 Gender, Male (%) 85 (65.9%) 7(41.2%) 25 (100%) 11 (55%) 0.328 Tumor location at presentation, n (%)Bladder 70 (54.3%) — — — — Ureter or 59 (45.7%) — — — — Renal pelvisPathologic stage, n (%) pTa/Tis/T1 63 (48.8%) — — — — pT2 30 (23.3%) — —— — pT3/T4 36 (27.9%) — — — — Pathological grade, n (%) Low 37 (28.7%) —— — — High 92 (71.3%) — — — — Tumor diameter, n (%) <1 cm 29 (22.5%) — —— — 1-3 cm 74 (57.4%) — — — — ≥3 cm 26 (20.1%) — — — — Number of tumors,n (%) Single 48 (37.2%) — — — — Multiple 81 (62.8%) — — — — SurgicalMethods TURBT 68 (52.7%) — — — — Cystectomy 15 (11.6%) — — — —Nephrouretectomy 46 (35.7%) — — — — Lymphovascular invasion, n (%) Total65 — — — — Positive 18 (27.7%) — — — — Negative 47 (72.3%) — — — — Lymphnode metastasis Positive 10 (7.8%) — — — — Negative 119 (92.2%) — — — —Chemotherapy Intravesical 69 (53.5%) — — — — Adjuvant 32 (24.8%) — — — —No 28 (21.7%) — — — — Surgical margin, n (%) Positive 7 (5.4%) — — — —Negative 122 (94.6%) — — — —

II. A Method for Validating an Existence of a Urinary Exosome of thePresent Disclosure 2.1 Steps of the Method for Validating the Existenceof the Urinary Exosome

FIG. 1 is a flow diagram showing a method 100 for validating anexistence of a urinary exosome according to one embodiment of thepresent disclosure. The method 100 for validating the existence of theurinary exosome includes a step 110, a step 120, a step 130 and a step140.

In the step 110, a sampling step is provided. First morning urinesamples are obtained from the subjects. For each urine sample (˜50 ml),one protease inhibitor cocktail tablet (Roche, Mannheim, Germany) isadded. The urine sample is centrifuged at 1,000×g for 10 minutes toremove debris. Then the urine sample is stored at −80° C. untilsubsequent serially centrifugation step or directly performed theserially centrifugation step.

In the step 120, a serially centrifugation step is performed on theurine sample, wherein the serially centrifugation step further includessteps as follows. Aforementioned urine sample is centrifuged at 17,000×gfor 10 minutes at 4° C. to obtain a first supernatant and a firstprecipitate. The first supernatant is collected in a new tube, and thefirst precipitate is resuspended with an isolation solution (10 mmtriethanolamine/250 mm sucrose, pH 7.6, and 0.5 mm PMSF) before adding200 mg/ml dithiotheritol to obtain a first mixture. The first mixture isincubated at 95° C. for 2 minutes, and then centrifuged at 17,000×g for30 minutes at 4° C. to obtain a second supernatant and a secondprecipitate. The first supernatant and the second supernatant are mixedto obtain a second mixture, and the second mixture is centrifuged at200,000×g for 1 hour at 4° C. to obtain a third precipitate.

In the step 130, an extraction step is provided. The third precipitateis resuspended with an extraction solvent to obtain a third mixture,wherein the extraction solvent is formic acid and/or acetonitrile.Further, the extraction solvent is the formic acid at a weightpercentage in a range from 50% to 98% or a mixture of the formic acid atthe weight percentage in the range from 25% to 50% and 50 weightpercentage of the acetonitrile. Then the third mixture is centrifuged at10,000×g for 15 minutes at 4° C. to remove insoluble sediment and obtaina fourth supernatant.

In the step 140, a mass spectrometric analysis step is performed on thefourth supernatant by using a matrix-assisted laser desorptionionization-time of flight mass spectrometry (MALDI-TOF MS), wherein theurinary exosome is existed in the fourth supernatant when at least twopolypeptides of a 3367 m/z polypeptide, a 3441 m/z polypeptide, a 3483m/z polypeptide and a 10884 m/z polypeptide are detected in the fourthsupernatant. The 3367 m/z polypeptide has amino acid sequence of SEQ IDNO:1, the 3441 m/z polypeptide has amino acid sequence of SEQ ID NO:2,the 3483 m/z polypeptide has amino acid sequence of SEQ ID NO:3, and the10884 m/z polypeptide has amino acid sequence of SEQ ID NO:4.

2.2 Confirmation of the Urinary Exosome

To confirm the urinary exosome can be isolated by the step 110 and thestep 120, expressions of biomarkers of the urinary exosome in the thirdprecipitate treated with the step 110 and the step 120 are analyzed by aWestern immunoblotting, and morphology of the urinary exosome isobserved by a transmission electron microscope.

The steps of the Western immunoblotting are as follows. The thirdprecipitate is lysed by RIPA lysis buffer (10 mM Tris-HCl, 1 mM EDTA, 1mM EGTA, 50 mM NaCl, 50 mM NaF, 20 mM Na₄P₂O₇, 1 mM Na₃VO₄, and 1%Triton X-100). The lysis is transferred to a microcentrifuge tube, andthen centrifuged at 12,000×g for 10 minutes to a supernatant. Theprotein concentration is determined by Bradford protein quantification.20 μg proteins are solubilized in Laemmli sample buffer (1.5% SDS, 6%glycerol, and 10 mM Tris-HCI (pH 6.8)), and then boiled at 100 ° C. for10 minutes. The proteins with different physical properties areseparated by a one-dimensional sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (1 D SDS-PAGE) and transferred electrophoretically ontopolyvinylidene fluoride (PVDF) membranes. After blocking the PVDFmembranes with 5% nonfat milk at room temperature for 1 hour, the PVDFmembranes are probed overnight at 4° C. with primary monoclonalantibodies to TSG101 (1:500) (Thermo Scientific), Alix (1:200)(chemicon) and actin (1:1000) (Rockland). The TSG101 and the Alix arethe biomarkers of the exosome, and the actin is an internal control.Thereafter, the PVDF membranes are probed with peroxidase-conjugatedanti-rabbit or anti-mouse IgG (1:10,000) for 1 hour at room temperature.Immunoreactivity is detected by enhanced chemiluminescence.

FIG. 2 is analytical results of the Western immunoblotting to detectbiomarkers of the urinary exosome of the subject. The (1) to (5)represent the urine samples obtained from different groups of thesubjects, wherein (1) is the group of the patients with low-gradeurothelial cancer, (2) is the group of the patients with high-gradeurothelial cancer, (3) is the group of the patients with the prostatecancer, (4) is the group of the patients with the urinary tractinfection, and (5) is the group of the healthy subjects. In FIG. 2, thethird precipitate isolated from different groups of the subjects can bedetected TSG101 expressions and Alix expressions. It indicates that theurinary exosome can be isolated by the step 110 and the step 120.

The third precipitate is further resuspended in 2.5% glutaraldehyde andapplied to Formvar-coated carbon-stabilized copper grids. The grids aredried at room temperature, washed twice with PBS, and then stained with2% (w/v) uranyl acetate for 10 minutes. The grid is examined with a JOEL200CX transmission electron microscope.

FIG. 3 is a set of micrographs of the transmission electron microscopeof the urinary exosome of the subject. Under the transmission electronmicroscope, round-shaped membranous vesicles with ˜50 nm to 100 nmdiameters are clearly observed in the third precipitate, which areconsistent with the morphology of the exosome. It indicates that theurinary exosome can be isolated by the step 110 and the step 120 indeed.

2.3 Comparison of the Results of the Urinary Exosome Extracted byDifferent Extraction Solvents

To test the best extraction conditions in the step 130, the thirdprecipitate is extracted by fourteen different extraction conditionsrespectively in this example. The extraction condition is (1) ddH₂O asthe extraction solvent, (2) 25 weight percentage of the acetonitrile asthe extraction solvent, (3) 50 weight percentage of the acetonitrile asthe extraction solvent, (4) 75 weight percentage of the acetonitrile asthe extraction solvent, (5) 25 weight percentage of the formic acid asthe extraction solvent, (6) 50 weight percentage of the formic acid asthe extraction solvent, (7) 75 weight percentage of the formic acid asthe extraction solvent, (8) 98 weight percentage of the formic acid asthe extraction solvent, (9) the mixture of 25 weight percentage of theformic acid with 50 weight percentage of the acetonitrile as theextraction solvent, (10) the mixture of 35 weight percentage of theformic acid with 50 weight percentage of the acetonitrile as theextraction solvent, (11) the mixture of 45 weight percentage of theformic acid with 50 weight percentage of the acetonitrile as theextraction solvent, (12) the mixture of 50 weight percentage of theformic acid with 50 weight percentage of the acetonitrile as theextraction solvent, (13) the RIPA lysis buffer as the extraction solventwith a sonication, and (14) sonication, respectively. Aforementionedextraction solvent is respectively added into the third precipitate toobtain the third mixture, and the third mixture is centrifuged at10,000×g for 15 minutes at 4° C. to remove the insoluble sediment andobtain the fourth supernatant. Each extraction condition is repeatedwith eight technical replicates.

The concentration of total proteins in the fourth supernatant obtainedfrom aforementioned fourteen different extraction conditions isdetermined by the Bradford protein quantification respectively. Thedifference among the eight technical replicates at each extractioncondition is compared by statistical software. The extracted proteinsare further analyzed by the 1D SDS-PAGE and a silver stain, wherein 12%SDS-polyacrylamide gel is used in the 1D SDS-PAGE. The electrophoresisis performed on a power supply set at 75 V/gel for the stacking gel and110 V/gel for the resolving gel. All gels are placed in fixing solution(methanol: acetic acid: deionized water=40:10:50) for 1 hour, thenwashed twice in ddH₂O for 10 minutes. The gels are stained with silverstain, and then analyzed bands of the gels.

FIG. 4A is the protein quantitative results according to one embodimentof the present disclosure. In FIG. 4A, there is no statisticaldifference among the eight technical replicates at each extractioncondition (p value is 0.99, determined by ANOVA test). High percentage(50% to 98%) of the formic acid or the mixture of 25% to 50% of theformic acid with 50% of the acetonitrile as the extraction solvent(extraction conditions (6)-(13)) yields similar protein concentration.Furthermore, the protein concentrations of the fourth supernatantobtained from the extraction conditions (6)-(13) are higher than thatobtained from the extraction conditions (1)-(5) and (14).

FIG. 4B is analytical result of sliver stain according to one embodimentof the present disclosure. In FIG. 4B, the extraction conditions(6)-(13) produce more prominent bands at both high molecular weights andlow molecular weights via the 1D SDS-PAGE separating the proteins withdifferent physical properties. It indicates that the proteins and theprotein concentration of the exosome extracted by the formic acid and/orthe acetonitrile in the present disclosure is similar to that extractedby the RIPA lysis buffer (the conventional method).

2.4 Biomarkers for Validating the Existence of the Urinary Exosome

The fourth supernatant obtained from aforementioned fourteen differentextraction conditions is further performed the mass spectrometricanalysis step by the MALDI-TOF MS respectively. The analyte solution ismixed with saturated sinapinic acid (SA) solution (30:70acetonitrile/0.1% trifluoroacetic acid) at volume ratios of 1:1 or 1:5.1 μL of the analyte/SA solution is placed on the MALDI-TOF target. Afteranalyte/SA co-crystallization, the sample plate is analyzed by MALDI-TOF(Ultraflex III TOF/TOF; Bruker Daltonics). The MALDI-TOF is operated inlinear positive ion mode with 25-kV accelerating voltage at a laserfrequency of 50 Hz with a mass range from 1000 to 23,000 Da.Peptide/protein calibrations are carried out using a peptide/proteincalibration standard kit (Bruker Daltonics). MALDI-TOF mass spectra areprocessed using flexAnalysis 3.0 software (Bruker Daltonics).

FIGS. 5A to 5D are the MALDI-TOF mass spectra according to oneembodiment of the present disclosure, wherein FIG. 5A are the MALDI-TOFmass spectra of the fourth supernatant obtained from extractionconditions (1)-(4), FIG. 5B are the MALDI-TOF mass spectra of the fourthsupernatant obtained from extraction conditions (5)-(7), FIG. 5C are theMALDI-TOF mass spectra of the fourth supernatant obtained fromextraction conditions (8)-(11), and FIG. 5D are the MALDI-TOF massspectra of the fourth supernatant obtained from extraction conditions(12)-(14). In FIGS. 5A to 5D, the fourth supernatant obtained from theextraction conditions (6)-(12) has similar MALDI-TOF mass spectrumproducing particular peaks at m/z 3367, m/z 3441, m/z 3483, m/z 5593,m/z 5947 and m/z 10884, which represents the 3367 m/z polypeptide 210,the 3441 m/z polypeptide 220, the 3483 m/z polypeptide 230, the 5593 m/zpolypeptide 250, the 5947 m/z polypeptide 260 and the 10884 m/zpolypeptide 240. However, because the extraction condition (13) uses theRIPA lysis buffer containing detergent and salts, the MALDI-TOF signalsare significantly impaired despite performing sample purification priorto MALDI-TOF analysis. Therefore, the MALDI-TOF mass spectrum of thefourth supernatant obtained from extraction condition (13) issignificant different from the MALDI-TOF mass spectra of the fourthsupernatant obtained from extraction conditions (6)-(12). 75% of theformic acid is selected as the extraction solvent for subsequentexamples.

To confirm that whether there are same particular polypeptides ordifferent particular polypeptides in different groups of the subjects,the MALDI-TOF mass spectra of the fourth supernatant extracted from theUC patients, the UTI patients, the PC patients and the healthy subjectsare compared by a two-dimensional (2-D) pseudo gel.

FIG. 6 is an analytical result of the 2-D pseudo gel according to oneembodiment of the present disclosure. In FIG. 6, the 3367 m/zpolypeptide 210, the 3441 m/z polypeptide 220, the 3483 m/z polypeptide230 and the 10884 m/z polypeptide 240 are consistently detected in theexosomes from the UC patients and the non-UC subjects. Therefore, the3367 m/z polypeptide 210, the 3441 m/z polypeptide 220, the 3483 m/zpolypeptide 230 and the 10884 m/z polypeptide 240 can act as thebiomarkers for identifying the urinary exosome. Furthermore, when atleast two polypeptides of the 3367 m/z polypeptide 210, the 3441 m/zpolypeptide 220, the 3483 m/z polypeptide 230 and the 10884 m/zpolypeptide 240 are detected, the urinary exosome is existed.

The sequence of the 3367 m/z polypeptide 210, the 3441 m/z polypeptide220, the 3483 m/z polypeptide 230 and the 10884 m/z polypeptide 240 arefurther identified in this example. The concentration of the totalproteins in the fourth supernatant is determined by the Bradford proteinquantification. The samples of the different groups are taken at fixedprotein amount. For higher separation resolution, electrophoresis on 4%to 12% Bis-Tris NuPAGE® gels (Invitrogen) is performed to purify andseparate the 3367 m/z polypeptide 210, the 3441 m/z polypeptide 220 andthe 3483 m/z polypeptide 230. The electrophoresis is performed at 75V/gel for the stacking gel and 110 V/gel for the resolving gel. Afterseparation, the gel is stained with Coomassie Brilliant Blue G250 anddestained by ddH₂O. Peptide bands ˜3000 Da are excised and extractedwith 50% acetonitrile/0.1% formic acid. The extracted peptides areanalyzed by the MALDI-TOF MS to confirm the presence of the 3367 m/zpolypeptide 210, the 3441 m/z polypeptide 220 and the 3483 m/zpolypeptide 230, and then identified by a nanoLC-MS/MS analysis. The3367 m/z polypeptide 210 is identified as a peptide fragment of theneutrophil defensin with the sequence referenced as SEQ ID NO: 1(monoisotopic mass: m/z 3365.7 Da; most abundant mass: m/z 3367.7 Da).The 3441 m/z polypeptide 220 is identified as the peptide fragment ofthe neutrophil defensin with the sequence referenced as SEQ ID NO: 2(monoisotopic mass: m/z 3439.9 Da; most abundant mass: m/z 3440.9 Da).The 3483 m/z polypeptide 230 is identified as the peptide fragment ofthe neutrophil defensin with the sequence referenced as SEQ ID NO: 3(monoisotopic mass: m/z 3481.8 Da; most abundant mass: m/z 3482.8 Da).The 10884 m/z polypeptide 240 is purified by a liquid chromatography(LC), digested with trypsin, and analyzed by the nano-LC MS/MS. The10884 m/z polypeptide 240 is identified as a large peptide fragment ofthe S100 A9 with the sequence referenced as SEQ ID NO: 4 (monoisotopicmass: m/z 10878.428 Da; most abundant mass: m/z 10884.443 Da).

III. A Method for Non-Invasive Method for Identifying an UrothelialCancer of the Present Disclosure

FIG. 7 is a flow diagram showing a non-invasive method 300 foridentifying an urothelial cancer according to another embodiment of thepresent disclosure. The non-invasive method 300 for identifying theurothelial cancer includes a step 310, a step 320, a step 330 and a step340.

In the step 310, the sampling step is provided. First morning urinesamples are obtained from the subjects. For each urine sample (˜50 ml),one protease inhibitor cocktail tablet (Roche, Mannheim, Germany) isadded. The urine sample is centrifuged at 1,000×g for 10 minutes toremove debris. Then the urine sample is stored at −80° C. untilsubsequent serially centrifugation step or directly performed theserially centrifugation step.

In the step 320, the serially centrifugation step is performed on theurine sample, wherein the serially centrifugation step further includessteps as follows. The urine sample is centrifuged at 17,000×g for 10minutes at 4° C. to obtain the first supernatant and the firstprecipitate. The first supernatant is collected in new tube, and thefirst precipitate is resuspended with the isolation solution (10 mmtriethanolamine/250 mm sucrose, pH 7.6, 0.5 mm PMSF) before adding 200mg/ml dithiotheritol to obtain the first mixture. The first mixture isincubated at 95° C. for 2 minutes, and then centrifuged at 17,000×g for30 minutes at 4° C. to obtain the second supernatant and the secondprecipitate. The first supernatant and the second supernatant are mixedto obtain the second mixture, and the second mixture is centrifuged at200,000×g for 1 hour at 4° C. to obtain the third precipitate.

In the step 330, the extraction step is provided. The third precipitateis resuspended with the extraction solvent to obtain the third mixture,wherein the extraction solvent is formic acid and/or acetonitrile.Further, the extraction solvent is the formic acid at the weightpercentage in the range from 50% to 98% or the mixture of the formicacid at the weight percentage in the range from 25% to 50% with 50weight percentage of the acetonitrile. Then the third mixture iscentrifuged at 10,000×g for 15 minutes at 4° C. to remove the insolublesediment and obtain the fourth supernatant.

In the step 340, the mass spectrometric analysis step is performed onthe fourth supernatant by using the MALDI-TOF MS to detect whether thefourth supernatant has a 5593 m/z polypeptide and/or a 5947 m/zpolypeptide 260. The 5593 m/z polypeptide has amino acid sequence of SEQID NO:5, and the 5947 m/z polypeptide 260 has amino acid sequence of SEQID NO:6.

3.1 The Biomarkers for Detecting the Urothelial Cancer

Referring back to FIG. 6, which is analytical result of the 2-D pseudogel according to one embodiment of the present disclosure. The 5593 m/zpolypeptide 250 and the 5947 m/z polypeptide 260 are specificallydetected in the UC patients compared to the non-UC subjects (the UTIpatients, the PC patients and the healthy subjects). Therefore, the 5593m/z polypeptide 250 and the 5947 m/z polypeptide 260 can act as thebiomarkers for identifying the urothelial cancer. The sensitivity andspecificity of the 5593 m/z polypeptide 250 alone for detecting theurothelial cancer are 50.4% and 96.9%, respectively, with an AUC (thearea under ROC curve) analyzed by ClinPro Tools 3.0 software (BrukerDaltonics) of 0.736. The sensitivity and specificity of the 5947 m/zpolypeptide 260 alone for detecting the urothelial cancer are 62.0% and92.3%, respectively, with the AUC of 0.772. The sensitivity andspecificity of the 5593 m/z polypeptide 250 and the 5947 m/z polypeptide260 combined for detecting the urothelial cancer are 62.70% and 87.59%,respectively, with the AUC of 0.87. Therefore, the 5593 m/z polypeptide250 and the 5947 m/z polypeptide 260 not only can be used alone as thebiomarker for detecting the urothelial cancer but also can be combinedused for detecting the urothelial cancer.

The 5593 m/z polypeptide 250 and the 5947 m/z polypeptide 260 arefurther purified by the LC, digested with trypsin, and analyzed by thenano-LC MS/MS. The 5593 m/z polypeptide 250 is identified as thefragment peptide of α-1-antitrypsin with the sequence referenced as SEQID NO: 5 (monoisotopic mass: m/z 5590.089 Da; most abundant mass: m/z5593.098 Da). The 5947 m/z polypeptide 260 is identified as the fragmentpeptide of the H2B1 K with the sequence referenced as SEQ ID NO: 6(monoisotopic mass: m/z 5944.107 Da; most abundant mass: m/z 5947.115Da).

3.2 Protein Expression Levels of Biomarkers of the Urothelial CancerDetected by an Immunohistochemical Staining

To confirm that the α-1 antitrypsin and the H2B1 K can indeed be used asthe biomarkers for detecting the urothelial cancer, theimmunohistochemical staining is further performed in this example.Tissue speciments of the immunohistochemical staining are obtained from122 UC patients and 26 healthy subjects.

The steps of the immunohistochemical staining are as follows. The tissuespeciments are initially formalin fixed and paraffine embedded. Allimmunohistochemical staining is carried out on a Leica Bond-Maxautostainer (Leica Microsystems) according to the manufacturer'sprotocol. Antigen retrieval is carried out at pH 8 with EpitopeRetrieval 2 solution (Leica Microsystems) for 20 minutes at 100° C. Thetissue speciments are then incubated for 15 hours at room temperaturewith the primary antibodies at the following dilutions: rabbitpolyclonal anti-trypsin (1:1600; Novocastra) and rabbit polyclonal H2B1K (1:400; NOVUS). The tissue speciments are conjugated with DABsecondary antibody to detect staining results. After the detection, thetissue speciments are then counterstained by hematoxylin.

In the immunohistochemical staining, the biomarker expression level isanalyzed by estimating a staining intensity of the biomarker of theurothelial carcinoma in 25 high power fields (magnification 400×), andit is classified as 0, 1, 2, and 3. A pathologist analyzes theimmunohistochemical stained tissue sections without knowing thediagnostic results during the histopathological diagnosis. Criteria ofthe classification are as follows: 0 represents that no cells arestained with color, 1 represents that the cells are stained with weakintensity of color on intact cell, 2 represents that the cells arestained with moderate intensity of color on intact cell, and 3represents that the cells are stained with strong intensity of color onintact cell. The stained cell number is estimated as a percentage(stained cell number/total cell number×100%), and a IHC score in theimmunohistochemical staining is calculated according to the followingformula I:

IHC score=Σ(i×Pi)   formula I,

wherein i represents the staining intensity of the biomarker (0, 1, 2,or 3), Pi represents the percentage of the stained cells (varied from 0%to 100%), and the IHC score ranges from 0 to 300.

FIG. 8 is a set of micrographs of the immunohistochemical staining,which includes hematoxylin and eosin staining (HE staining) results inhigh-grade and low-grade UC tissue specimens and the IHC stainingresults of the a-1 antitrypsin and the H2B1 K in the high-grade andlow-grade UC tissue specimens. In FIG. 8, the α-1 antitrypsin iscytoplasmic staining, while the H2B1 K is nuclear staining. In bothhigh-grade and low-grade UC tissue specimens, the staining intensity ofthe α-1 antitrypsin and the H2B1 K in the UC tissue specimen is strongerthan neighboring normal uroepithelium.

When comparing the IHC staining results of the UC tissue specimen withthat of the non-UC tissue specimen, the sensitivity/specificity of theα-1 antitrypsin and the H2B1 K expression in the UC tissues specimen are35.2%/96.2%, and 30.8%/96.7%, respectively.

FIG. 9A is the quantification of the IHC score of the α-1 antitrypsinexpressions in the non-UC tissue specimens and the UC tissues specimensdetected by the immunohistochemical staining. FIG. 9B is thequantification of IHC score of H2B1 K expressions in the non-UC tissuespecimens and the UC tissues specimens detected by theimmunohistochemical staining. FIG. 10A is the quantification of IHCscore of the α-1 antitrypsin expressions in high-grade UC tissuespecimens and low-grade UC tissue specimens detected by theimmunohistochemical staining. FIG. 10B is the quantification of IHCscore of the H2B1 K antitrypsin expressions in high-grade UC tissuespecimens and low-grade UC tissue specimens detected by theimmunohistochemical staining.

In FIGS. 9A and 9B, the IHC scores for the α-1 antitrypsin are 4.40-foldhigher significantly in the UC tissue specimens than in the non-UCtissue specimens (p value is 0.038). For the H2B1 K expressions, the IHCscore of the UC tissue specimens is up to 8.20-fold higher compared withthe non-UC tissue specimens (p value is 0.005). In FIGS. 10A and 10B,the α-1 antitrypsin is highly expressed with 6.32-fold change in highgrade UC tissue specimens than low grade UC tissue specimens (p value is0.005); the expression of the H2B1 K is 2.75-fold stronger in low gradeUC tissue specimens compared with high grade (p value is 0.037).

Table 2 is correlations of the IHC socre of the α-1 antitrypsin and theH2B1 K with clinical parameters of the subjects.

TABLE 2 The correlations of the IHC socre of the α-1 antitrypsin and theH2B1K with clinical parameters of the subjects Normal versus UC Normal(26) UC (122) P value α-1 antitrypsin 55.48 78.55 0.002* H2B1K 54.1378.84 0.007* Gender Female (43) Male (79) P value α-1 antitrypsin 53.9365.62 0.269 H2B1K 65.59 59.27 0.079 Age <65 (19) ≥65 (103) P value α-1antitrypsin 54.89 62.72 0.299 H2B1K 57.47 62.24 0.587 Pathological gradeof the urothelial cancer Low (46) High (76) P value α-1 antitrypsin51.73 67.41 0.005* H2B1K 70.01 56.35 0.037* Tumor size <1 cm (61) 1-3 cm(43) ≥3 cm (18) P value α-1 antitrypsin 53.01 60.78 92 <0.001* H2B1K62.28 62.29 56.03 0.773 Number of tumors Single (72) Multiple (49) Pvalue α-1 antitrypsin 64.41 55.99 0.129 H2B1K 61.17 60.74 0.947 Tumorinvasion degree (T) Ta + Tis T1 + T2 T3 + T4 P value α-1 antitrypsin49.88 62.94 61.48 0.512 H2B1K 94.25 56.13 63.43 0.013* Lymph nodemetastasis degree (N) N0 (117) N1 (2) N2 (2) P value α-1 antitrypsin59.97 70.25 111.75 0.046 H2B1K 60.75 70.5 66.0 0.907 Distant metastasisdegree (M) M0 (117) M1 (5) P value α-1 antitrypsin 60.43 86.6 0.057H2B1K 62.9 28.7 0.033* Pathologic stage 0 (7) I + II (64) III (38) IV(13) P value α-1 antitrypsin 51.29 61.13 56.07 84.69 0.021* H2B1K 95.6458.06 62.01 58.54 0.062

In Table 2, the α-1 antitrypsin is significantly positively correlatedwith the pathological grade of the UC (p value is 0.005), the tumor size(p value is less than 0.001), and the pathologic stage (p value is0.021). In term of the tumor size, the tissue specimens from tumorsize >3 cm have the highest IHC scores of the α-1 antitrypsin, thentumor size 1-3 cm, and then tumor size <1 cm (p value is less than0.001). The IHC scores of the α-1 antitrypsin are associated withpathologic stage of the UC. Stage 4 UC have the highest IHC expressionscores of the α-1 antitrypsin then other IHC scores of other stages.

The results of the immunohistochemical staining further validate thatthe 5593 m/z polypeptide 250 and the 5947 m/z polypeptide 260 can beused as the biomarkers for detecting the urothelial cancer.

IV. A Method for Predicting a Recurrence and a Progression of anUrothelial Cancer Patient after a Treatment of the Present Disclosure

FIG. 11 is a flow diagram showing a method 500 for predicting therecurrence and the progression of the urothelial cancer patient afterthe treatment according to yet another embodiment of the presentdisclosure. The method 500 for predicting the recurrence and theprogression of the urothelial cancer patient after the treatmentincludes a step 510, a step 520, a step 530 and a step 540.

In the step 510, a sampling step is provided. First morning urinesamples are obtained from the subjects. For each urine sample (˜50 ml),one protease inhibitor cocktail tablet (Roche, Mannheim, Germany) isadded. The urine sample is centrifuged at 1,000×g for 10 minutes toremove debris. Then the urine sample is stored at −80° C. untilsubsequent serially centrifugation step or directly performed theserially centrifugation step.

In the step 520, the serially centrifugation step is performed on theurine sample, wherein the serially centrifugation step further includessteps as follows. The urine sample is centrifuged at 17,000×g for 10minutes at 4° C. to obtain the first supernatant and the firstprecipitate. The first supernatant is collected in new tube, and thefirst precipitate is resuspended with the isolation solution (10 mmtriethanolamine/250 mm sucrose, pH 7.6, 0.5 mm PMSF) before adding 200mg/ml dithiotheritol to obtain the first mixture. The first mixture isincubated at 95° C. for 2 minutes, and then centrifuged at 17,000×g for30 minutes at 4° C. to obtain the second supernatant and the secondprecipitate. The first supernatant and the second supernatant are mixedto obtain the second mixture, and the second mixture is centrifuged at200,000×g for 1 hour at 4° C. to obtain the third precipitate.

In the step 530, the extraction step is provided. The third precipitateis resuspended with the extraction solvent to obtain the third mixture,wherein the extraction solvent is formic acid and/or acetonitrile.Further, the extraction solvent is the formic acid at the weightpercentage in the range from 50% to 98% or the mixture of the formicacid at the weight percentage in the range from 25% to 50% with 50weight percentage of the acetonitrile. Then the third mixture iscentrifuged at 10,000×g for 15 minutes at 4° C. to remove the insolublesediment and obtain the fourth supernatant.

In the step 540, the mass spectrometric analysis step is performed onthe fourth supernatant by using the MALDI-TOF MS, wherein the urothelialcancer patient is determined to have a high recurrence and a highprogression after the treatment when the 5947 m/z polypeptide 260 isdetected in the fourth supernatant. The 5947 m/z polypeptide 260 hasamino acid sequence of SEQ ID NO:6.

4.1 Probability Analysis of Overall Recurrence-Free and OverallProgression-Free

In this example, 129 US patients receiving the TURBT, a cystectomy, or anephroureterectomy are classified into the UC patient with positive 5947m/z polypeptide expression (m/z 5947 (+)) and negative 5947 m/zpolypeptide expression (m/z 5947 (−)) according to the expression levelof the biomarker. Statistical analysis is further performed by usingSPSS 20.0 (SPSS Inc.). A survival time is analyzed by Kaplan-Meierestimate, and a statistical significant of the survival time is furtheranalyzed by a log rank test. A p value of <0.05 is consideredstatistically significant in all statistical analysis of this example.

A survival analysis is a common statistical analysis of clinical trialsin various cancers, and is a statistic that deals with time variable. Inmore details, the survival analysis deals with analysis of time durationuntil one or more events happen. Two evaluation indexes of the timevariable, an overall recurrence-free and an overall progression-free,are used in this example. The event is “the recurrence or a death” inthe evaluation index of the overall recurrence-free, and a length oftime from a subject participating in the clinical trial until therecurrence is then observed. The event is “the progression or the death”in the evaluation index of the overall progression-free, and the lengthof time from the subject participating in the clinical trial until theprogression is then observed. A median follow-up time is 13.3 months(range, 2-32 months). At the time of analysis, the UC recurred in 80patients (62.0%), and four patients died (3.1%). Among the deceasedpatients, three died of metastatic UC.

FIG. 12A is a survival curve showing overall recurrence-free accordingto yet another embodiment of the present disclosure. FIG. 12B is thesurvival curve showing overall progression-free according to yet anotherembodiment of the present disclosure. A ladder in the figure representsa time point in an occurrence of the event, and a cross-mark in thefigure represents the time point in the occurrence of censored data. InFIG. 12A, an overall recurrence-free time in the UC patient withnegative 5947 m/z polypeptide expression is longer than that in the UCpatient with positive 5947 m/z polypeptide expression. The p valueanalyzed by the log rank test is less than 0.001 between the UC patientwith positive 5947 m/z polypeptide expression and the UC patient withnegative 5947 m/z polypeptide expression. In FIG. 12B, an overallprogression-free time in the UC patient with negative 5947 m/zpolypeptide expression is longer than that in the UC patient withpositive 5947 m/z polypeptide expression. The p value analyzed by thelog rank test is 0.018 between the UC patient with positive 5947 m/zpolypeptide expression and the UC patient with negative 5947 m/zpolypeptide expression. These results indicate that the 5947 m/zpolypeptide 260 has an important effect on predictions of the recurrenceand the progression of the UC patient after the treatment.

4.2 Treatment Effect Assessment Analysis

The log rank test is only used to determine whether the differences aresignificant between different groups, but it can not estimate atreatment effect. Therefore, a Cox proportional hazard model is used forestimating the treatment effect according dependent variables in thisexample. There are two assays, an univariate regression assay and anmultivariate regression assay, are performed, wherein the univariateregression assay includes one independent variable, and the multivariateregression assay includes at least two independent variables.

Table 3 is the treatment effect assessment analysis of the recurrence.When the hazard ratio is greater than 1, it indicates that thepercentage of a death occurrence in the patient having the independentvariable is greater than that in the patient who does not have theindependent variable. Furthermore, when the HR is greater than 1 and thep value is less than 0.05, it indicates that the independent variablehas a statistically significant difference. The univariate regressionassay indicates that the 5947 m/z polypeptide 260 is associated with ahigher risk of the recurrence of the UC (HR, 2.36; p value is 0.001).The multivariate regression assay shows that the 5947 m/z polypeptide260 is an independent predictor of the UC recurrence (HR, 2.29; p valueis 0.001). It indicates that the occurrence of the recurrence in thepatient with positive 5947 m/z polypeptide expression is 2.29 timeshigher than that in the patient with negative 5947 m/z polypeptideexpression. The results in Table 3 show that the 5947 m/z polypeptide260 has the important effect on the prediction of the recurrence of theUC patient after the treatment.

TABLE 3 The treatment effect assessment analysis of the recurrenceUnivariate analysis Multivariate analysis Predictors HR P value HP Pvalue Age (≥65 yrs vs ≤ 65 yrs) 1.79 0.021 1.71 0.034** Gender: males vsfemale 1.003 0.99 1.089 0.722 Tumor size 1-3 cm vs < 1 cm 1.194 0.5351.31 0.371 ≥3 cm vs < 1 cm 1.022 0.933 1.11 0.794 Grade: High vs Low1.08 0.754 1.03 0.909 Stage T2 vs Tis/Ta/T1 1.52 0.157 1.346 0.3 T3/T4vs T2 1.72 0.75 1.249 0.41 Numbers of tumors: 1.10 0.678 1.10 0.644multiple vs single Lymph node status: metastatsis 2.058 0.16 1.923 0.206Lymphovascular invasion: 1.196 0.609 1.017 0.963 positive Chemotherapy*:Yes, no 1.16 0.501 1.01 0.965 Surgical margin: positive vs 1.37 0.4931.14 0.774 negative 5947 m/z polypeptide: positive 2.36 0.001 2.290.001** 5593 m/z polypeptide: positive 1.54 0.647 1.27 0.289

Table 4 is the treatment effect assessment analysis of the progression.The univariate regression assay indicates that the 5947 m/z polypeptide260 is associated with the higher risk of the progression of the UC (HR,2.77; p value is 0.025). The multivariate regression assay shows thatthe 5947 m/z polypeptide 260 is the independent predictor of theprogression of the UC (HR, 3.11; p value is 0.039). It indicates thatthe occurrence of the progression in the patient with positive 5947 m/zpolypeptide expression is 3.11 times higher than that in the patientwith negative 5947 m/z polypeptide expression. The results in Table 4show that the 5947 m/z polypeptide 260 has the important effect on theprediction of the progression of the UC patient after the treatment.

TABLE 4 The treatment effect assessment analysis of the progressionUnivariate analysis Multivariate analysis Predictors HR P value HP Pvalue Age (≥65 yrs vs ≤ 65 yrs) 2.35 0.046 2.35 0.096 Gender: males vsfemale 1.048 0.899 1.036 0.481 Tumor size 1-3 cm vs < 1 cm 5.59 0.025.15 0.031 ≥3 cm vs < 1 cm 4.67 0.05 1.93 0.46 Grade: High vs Low 2.630.027 2.53 0.034 Stage T2 vs Tis/Ta/T1 1.08 0.867 1.07 0.893 T3/T4 vs T21.18 0.686 1.17 0.385 Numbers of tumors: 5.58 0.02 1.568 0.367 multiplevs single Lymph node status: metastatsis 1.23 0.776 1.079 0.47Lymphovascular invasion: 1.71 0.46 1.58 0.46 positive Chemotherapy*:Yes, no 1.54 0.227 1.42 0.338 Surgical margin: positive vs 7.58 <0.0014.13 0.031 negative 5947 m/z polypeptide: positive 2.77 0.025 3.110.039** 5593 m/z polypeptide: positive 1.156 0.683 1.222 0.649

To sum up, the method for validating the existence of the urinaryexosome is provided in the present disclosure, which uses a novelextraction method to obtain an extract. The extract can be directlyperformed the mass spectrometric analysis step to detect the 3367 m/zpolypeptide, the 3441 m/z polypeptide, the 3483 m/z polypeptide and the10884 m/z polypeptide. When at least two polypeptides of the 3367 m/zpolypeptide, the 3441 m/z polypeptide, the 3483 m/z polypeptide and the10884 m/z polypeptide are detected in the extract, the urinary exosomeis existed in the extract. It provides a platform for rapidly validatingthe existence of the urinary exosome, thus it can be a rapid diagnostictest for clinical and basic research. The non-invasive method foridentifying an urothelial cancer is also provided in the presentdisclosure, which uses the novel extraction method to obtain the extractcontaining the urinary exosome. The extract can be directly performedthe mass spectrometric analysis step to detect the 5593 m/z polypeptideand the 5947 m/z polypeptide. It provides a rapid and non-invasivemethod for quickly and objectively detecting whether the subjectsuffering from the urothelial cancer. The method for predicting therecurrence and the progression of the urothelial cancer patient afterthe treatment is further provided in the present disclosure, which usesthe novel extraction method to obtain the extract containing the urinaryexosome. The extract can be directly performed the mass spectrometricanalysis step to detect the 5947 m/z polypeptide. It provides thenon-invasive method for applying on a high-risk subjects with suspectedurothelial cancer. Therefore, the physician can determine whether the UCpatient need to treat with other drugs or other treatment plans toenhance the overall survival time and the disease free survival time inthe clinical application.

Although the present disclosure has been described in considerabledetail with reference to certain embodiments thereof, other embodimentsare possible. Therefore, the spirit and scope of the appended claimsshould not be limited to the description of the embodiments containedherein.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the structure of the presentdisclosure without departing from the scope or spirit of the disclosure.In view of the foregoing, it is intended that the present disclosurecover modifications and variations of this disclosure provided they fallwithin the scope of the following claims.

What is claimed is:
 1. A method for predicting a recurrence and aprogression of an urothelial cancer patient after a treatment,comprising: providing a sampling step, wherein a urine sample isobtained from a subject; performing a serially centrifugation step onthe urine sample, comprising: centrifuging the urine sample to obtain afirst supernatant and a first precipitate; resuspending the firstprecipitate with an isolation solution to obtain a first mixture, andcentrifuging the first mixture to obtain a second supernatant and asecond precipitate; and mixing the first supernatant and the secondsupernatant to obtain a second mixture, and centrifuging the secondmixture to obtain a third precipitate; providing an extraction step,wherein the third precipitate is resuspended with an extraction solventto obtain a third mixture, the third mixture is centrifuged to obtain afourth supernatant, and the extraction solvent is formic acid and/oracetonitrile; and performing a mass spectrometric analysis step on thefourth supernatant by using a matrix-assisted laser desorptionionization-time of flight mass spectrometry (MALDI-TOF MS); wherein theurothelial cancer patient is determined to have a high recurrence and ahigh progression after the treatment when a 5947 m/z polypeptide isdetected in the fourth supernatant.
 2. The method for predicting therecurrence and the progression of the urothelial cancer patient afterthe treatment of claim 1, wherein the extraction solvent is the formicacid at a weight percentage in a range from 50% to 98%.
 3. The methodfor predicting the recurrence and the progression of the urothelialcancer patient after the treatment of claim 2, wherein the extractionsolvent is 75 weight percentage of the formic acid.
 4. The method forpredicting the recurrence and the progression of the urothelial cancerpatient after the treatment of claim 1, wherein the extraction solventis a mixture of the formic acid at the weight percentage in the rangefrom 25% to 50% with 50 weight percentage of the acetonitrile.
 5. Themethod for predicting the recurrence and the progression of theurothelial cancer patient after the treatment of claim 1, wherein the5947 m/z polypeptide has amino acid sequence of SEQ ID NO:6.